Abstract Accurate segregation of chromosomes to daughter cells during cell division is critical for cell viability and normal development. Errors in chromosome segregation are correlated with cancer progression and cause some types of severe birth defects (e.g. Down Syndrome). Chromosome segregation is controlled by the mitotic/meiotic spindle, which is a large macromolecular structure composed primarily of microtubules and associated proteins. Chromosome movements are powered by attachment of microtubules to the kinetochore and errors in kinetochore-microtubule attachment lead to aneuploidy. Decades of work have identified many proteins that are important for the assembly and function of the mitotic spindle, yet much remains unknown about regulation of spindle assembly. Recent work from our lab and others has shown that in addition to proteins hundreds of different mRNAs and components of the translation apparatus as well as long noncoding RNAs are also localized to the mitotic spindle and chromosomes and play a role in kinetochore and spindle assembly. Our group and others have also recently demonstrated that centromeres are transcribed into a long, noncoding RNA important for kinetochore assembly and function. We showed that lncRNAs bind to and activate the mitotic kinase Aurora-B. In this proposal we describe a series of experiments designed to elucidate the roles and mechanisms of RNAs localized to the spindle and chromosomes during mitosis. We propose a series of experiments to examine how kinetochore proteins promote transcription of centromeric repeats during mitosis and how this is related to changes in kinetochore-microtubule attachment. We then propose a series of detailed biochemical experiments to understand how various centromere proteins interact with RNA and how this interaction influences their centromere functions. In addition to being regulated by RNA binding recent work demonstrates that Aurora-B controls the binding of noncoding RNAs to the chromosomes during mitosis. In the final Aim we propose to study the mechanism by which Aurora-B promotes the release of lncRNAs from mitotic chromosomes. We focus on the interaction between Aurora-B and hnrnp U and the consequences of a failure to remove lncRNAs from chromosomes during mitosis. Taken together, these Aims will provide insight into the interaction of Aurora-B with spindle, chromatin, and kinetochore-localized RNAs and how these interactions lead to successful chromosome segregation. These studies are likely to provide fundamental insight into the mechanisms of spindle assembly and cell division and could provide additional insight into mechanisms of cancer progression and the cause of birth defects.